A biophysical characterization of the iron coordination environment in wheat germ peroxidase

 The heme protein wheat germ peroxidase (isoenzyme C₂) and its cyanide-inhibited form have been investigated by means of electronic, CD and paramagnetic NMR spectroscopy. The data indicate a protein environment of the active site distinct from that of horseradish peroxidase (HRP), with a larger solvent accessibility. The iron is pentacoordinated at neutral and low pH, whereas a hydroxyl anion may be bound at alkaline pH. The fifth axial ligand is a His residue with a partial anionic character, as found in other peroxidases. A spin equilibrium is observed at high enzyme concentrations. Received: 17 September 1996 / Accepted: 10 January 1997     

Stress and asymmetry during arrested development of the Australian sheep blowfly.

The dieldrin and diazinon resistance systems of the Australian sheep blowfly (Lucilia cuprina) have been used previously to relate stress, departures from bilateral symmetry, developmental stability and relative fitness. These systems are now used to consider stress and asymmetry in a developmental context. Larval to adult development is shown to be significantly impaired after arrested development at 8 degrees C, however the asymmetry score of adults of a given genotype is similar after arrested or continuous development. Selection against dieldrin-resistant and unmodified diazinon-resistant genotypes occurs during arrested development because greater proportions of these genotypes pupae at 8 degrees C than do susceptible or modified diazinon-resistant genotypes. Pre-pupae of all genotypes complete development equally successfully when transferred from 8 degrees C to 27 degrees C. Adults fail to emerge when pupae formed at 8 degrees C undergo this temperature transition. Temperature-shift experiments show the asymmetry score is determined between pre-pupal and pupal stages of the life cycle. This stage occurs at 27 degrees C in arrested and continuously developing cultures providing an explanation for the independence of stress, selective mortality during developmental arrest and asymmetry score. The results emphasize the need for genetic, environmental and developmental data before an asymmetry phenotype can be directly related to developmental stability and relative fitness.     

The QUEST system for quantitative analysis of two-dimensional gels

The strategies and methods used by the QUEST system for two-dimensional gel analysis are described, and the performance of the system is evaluated. Radiolabeled proteins, resolved on two-dimensional gels and detected using calibrated exposures to film, are quantified in units of disintegrations per minute or as a fraction of the total protein radioactivity applied to the gel. Spot quantitation and resolution of overlapping spots is performed by two-dimensional gaussian fitting. Pattern matching is carried out for groups of gels called matchsets, and within each matchset every gel is matched to every other gel. During the matching process, spots are automatically added to each pattern at positions where unmatched spots were detected in other patterns. This results in enhanced accuracy for both spot detection and for matching. The spot fitting procedure is repeated after matching. Tests show that up to 97% of spots in each pattern can be matched and that fewer than 1% of the spots are matched inconsistently. Approximately 2000 proteins are detected from typical gels. Of these 1600 are high quality spots. Tests to measure the coefficient of variation of spot quantitation versus spot quality show that the average coefficient of variation for high quality spots is 21%. The intensities of the detected proteins range from 4 to 20,000 ppm of total protein synthesis. The QUEST analysis system has been used to build a quantitative database for the proteins of normal and transformed REF52 cells, as presented in the accompanying reports (Garrels, J., and Franza, B. R., Jr. (1989) J. Biol. Chem. 264, 5283-5298, 5299-5312).     

Metmyoglobin promotes arachidonic acid peroxidation at acid pH

The ability of metmyoglobin and other heme proteins to promote peroxidation of arachidonic acid under acidic conditions was investigated. Incubation of metmyoglobin with arachidonic acid resulted in a pH-dependent increase in lipid peroxidation as measured by the formation of thiobarbituric acid reactive products and oxygen consumption. Increased peroxidation was observed at pH levels below 6.0, reaching a plateau between pH 5.5 and 5.0. At comparable heme concentrations, metmyoglobin was more efficient than oxymyoglobin, methemoglobin, or ferricytochrome c in promoting arachidonic acid peroxidation. Metmyoglobin also promoted peroxidation of 1-palmityl-2-arachidonyl phosphatidylcholine and methylarachidonate but at significantly lower rates than arachidonic acid. Addition of fatty acid-free albumin inhibited arachidonic acid peroxidation in a molar ratio of 6 to 1 (arachidonic acid:albumin). Both ionic and non-ionic detergents inhibited metmyoglobin-dependent arachidonic acid peroxidation under acidic conditions. The anti-oxidants butylated hydroxytoluene and nordihydroguaiaretic acid and low molecular weight compounds with reduced sulfhydryl groups inhibited the reaction. However, mannitol, benzoic acid, and deferoxamine were without significant effect. Visible absorption spectra of metmyoglobin following reaction with arachidonic acid showed minimal changes consistent with a low level of degradation of the heme protein during the reaction. These observations support the hypothesis that metmyoglobin and other heme proteins can promote significant peroxidation of unsaturated fatty acids under conditions of mildly acidic pH such as may occur at sites of inflammation and during myocardial ischemia and reperfusion. This may be the result of enhanced aggregation of the fatty acid and/or interaction of the fatty acid with heme under acidic conditions.     

Eosinophils preferentially use bromide to generate halogenating agents

Human eosinophils preferentially utilize bromide to generate a brominating agent, even at physiological halide concentrations, where chloride (140 mM) is over 1000-fold greater than bromide (20-100 microM). Under the same conditions, neutrophils use chloride to generate a chlorinating agent. The total amount of active halogen trapped by 1,3,5-trimethoxybenzene from eosinophils increases by over 2-fold as the added bromide concentration increases from 0 to 100 microM, with approximately 40 nmol of halogen trapped per million cells at the highest bromide level. At least 25-35% of the oxygen consumed by stimulated eosinophils is directed toward the generation of halogenating species. Since the relative halogenating behavior of eosinophil peroxidase and neutrophil myeloperoxidase in this bromide range is essentially identical to that of the cells, the specificity of eosinophils toward bromide is intrinsic to eosinophil peroxidase and not to any special cellular properties. These results suggest that human eosinophils use bromide in vivo and that a deficiency of bromide may influence their ability to produce halogenating agents.     

Uracil-DNA glycosylase in insects. Drosophila and the locust

It has been reported that Drosophila lacks a uracil-DNA glycosylase but that a direct incising activity on uracil-containing DNA appeared developmentally only in third instar larvae. In contrast we have found by two independent assays, that uracil-DNA glycosylase exists in both Drosophila eggs as well as in third instar larvae. The first assay shows the liberation of [3H] uracil from a d(AT)n polymer randomly substituted with [3H]uracil by its synthesis in the presence of [3H] dUTP. The second fluorometric assay for uracil-DNA glycosylase depends on the unique topological properties of circular DNAs and has the advantage of detecting apyrimidinic/apurinic (AP) endonuclease activity as well. To test one other insect, locust eggs were also assayed for uracil-DNA glycosylase. The amount of uracil-DNA glycosylase correlated well with the amount of DNA in actively replicating cells.